Purification and characterisation of amphiphilic lactase/phlorizin hydrolase from human small intestine.
Human intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purification factor was approximately 600 and the recovery 14%. The enzyme was essentially free from other known brush-border peptidases and disaccharidases and appeared homogeneous in crossed immunoelectrophoresis and polyacrylamide gel electrophoresis in sodium dodecylsulphate. The purified enzyme hydrolyzed lactose (pH optimum 5.8--6.0, Km 21 mM), phlorizin (Km 0.44mM) and other beta-galactosides and beta-glucosides. Tris inhibited the hydrolysis of lactose whereas phlorizin hydrolysis was almost unaffected. The activity against these two substrates also showed different thermal stability. It is suggested that the human enzyme has two different sites: one for lactose hydrolysis, inhibited by phlorizin and one for phlorizin hydrolysis. By gel filtration on Ultrogel AcA 34 the amphiphilic form of the enzyme had a molecular weight of 320000 while the hydrophilic form (papain-treated) had a molecular weight of 280000. This indicates that the anchoring segment(s) plus the bound detergent has a molecular weight of approximately 40000. In polyacrylamide gel electrophoresis in sodium dodecylsulphate the fully denatured enzyme had an apparent molecular weight of 160000. It is therefore suggested that the human lactase/phlorizin hydrolase is composed of two monomers each with a molecular weight of 160000. The electromicroscopic picture gives further evidence for this suggestion. In addition the possibility of a high molecular weight, one polypeptide chain is discussed.
D004355:Drug Stability; D005696:Galactosidases / Q000302:isolation & purification*; D005959:Glucosidases / Q000302:isolation & purification*; D006801:Humans; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D010694:Lactase-Phlorizin Hydrolase / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism
D005696:Galactosidases; D005959:Glucosidases; D001616:beta-Galactosidase; D010694:Lactase-Phlorizin Hydrolase